Cellobiose is a disaccharide produced through the hydrolyzation process of cellulose widely distributed in, for instance, plants and has such a structure that two glucose molecules are linked through β-1,4 bond. Cellobiose is a naturally occurring material present in, for instance, stems of maize and pine needles and has a sweet taste, but it is not hydrolyzed by human. Accordingly, if cellobiose may efficiently and economically be prepared, it would be considered to be useful as a sweetening agent for use in health foods and foods for patients suffering from diabetes or a raw material for preparing cosmetic products and medicines.
As a conventionally known method for preparing cellobiose, there have been proposed those in which cellulose is chemically or enzymatically hydrolyzed.
Among them, examples of such enzymatic hydrolyzation methods include those comprising reacting cellulose with commercially available cellulase formulations containing cellulase originated from, for instance, microorganisms such as those belonging to the genus Trichoderma and Aspergillus to thus give cellobiose, as well as a method in which cellobiose is prepared using enzyme formulations from which β-glucosidase has been removed, while making use of the difference in the adsorbing power of cellulase to cellulose (Patent Document 1) and a method in which the hydrolysis reaction of cellulose is carried out in the presence of lignin (Patent Document 2). Moreover, there has also been known a method for the purification of cellobiose as an improved technique of the foregoing ones, which comprises hydrolyzing lignocellulose in the presence of a cellulase and a lignin-hydrolyzing bacterium or a lignin-hydrolyzing enzyme (Patent Document 3). However, the sugar solutions prepared through the hydrolysis according to these methods contain, in themselves, a large quantity of monosaccharides such as glucose and cello-oligosaccharides and accordingly, they cannot be considered to be highly pure cellobiose and the methods are likewise unsatisfied in the yields of cellobiose. In addition, if the content of glucose which is mixed in the sugar solution increases, it is expected that cellobiose is hardly crystallized.
On the other hand, as the foregoing chemical hydrolyzation techniques, there has been known, for instance, a method in which cellobiose is fractionated and isolated through the use of, for instance, a carbon column (Non-patent Document 1), but this method requires the use of quite complicated operations, including the use of a carbon column having a large volume and a large amount of ethanol for the elution from the column. Moreover, the method does not provide cellobiose in a satisfactorily high yield, it accordingly leads to a considerable increase in the production cost of cellobiose and the resulting product cannot accordingly be used in the field of foods. In addition, cellobiose has not yet been mass-produced industrially and accordingly, it has simply been prepared in a very small scale only for a reagent. There has recently been developed a method for preparing cellobiose (Patent Document 4), which comprises the steps of cooking and digesting a raw material containing naturally occurring lignocellulose and then partially hydrolyzing wet pulp thus obtained without using any drying step, by the action of a cellulase. This method comprises the steps of acting a cellulase on un-dried wet pulp which is quite susceptible to the action of the cellulase as a substrate, in a hydrolysis system equipped with an ultrafiltration membrane and continuously removing the hydrolyzation products from the reaction system through filtration and the method has thus been successful in the production of a cellobiose-containing sugar solution in a high yield. In addition, there has likewise been proposed a method for purifying cellobiose according to the same principle used in the foregoing method while using, as a raw material, bleached slush pulp (Patent Document 5).
However, the sugar solutions prepared according to these methods have high contents of glucose, oligosaccharides and other impurities and therefore, they cannot be used for the preparation of high purity cellobiose without using any further treatment.    Patent Document 1: JP-A-63-226294;    Patent Document 2: JP-A-05-317073;    Patent Document 3: JP-A-08-89274    Patent Document 4: JP-A-07-184678    Patent Document 5: JP-A-09-107987    Non-Patent Document 1: Miller, g. L., Methods in Carbohydrate Chemistry III, 1963, p. 134 (Academic Press).